In this post-genomics era, the development of a convenient and robust method for detection and quantitation of low abundance proteins and for analysis of protein-protein interactions is becoming critically important in the fields of biological and biomedical research, clinical diagnostics, environmental and food monitoring, and biotech-pharmaceutical industries (Duncan et al 2005, Srinivas et al 2002, and Albala 2001).
In addition, in the era, the development of a convenient and robust method for cell-based and tissue-based biomarker detection and quantitation is critically important in basic and clinical medicine, biomedical research, and biotech and pharmaceutical industry.
Traditionally sandwich-ELISA is a popular immunoassay to detect and quantitate antigen in biological and non-biological samples (Zangar et al, 2006, Zhou et al 2005). Its applications in clinical diagnostics, bio-medical research, food and environmental monitoring, and biotech and pharmaceutical industries are depended on its advantages: simplicity, sensitivity, and specificity.
Sandwich-ELISA uses specific antigen-antibody affinity binding to detect its antigen (Voller et al 1978, Lequin 2005, and Zangar et al 2006). The basic requirements for the method to detect antigen include having an antigen with at least two binding sites for specific antigen antibody binding reactions, having a pair of matched capture and detection antibodies which can be used to form a sandwich-like complex with the antigen for the antigen's capture and detection, and having a capture antibody with capacity to maintain its antigen binding activity after immobilized on a solid phase. The general procedure for the antigen detection includes that the antigen first reacts with excess solid-phase antibody (the capture antibody) immobilized on a solid phase, after incubation and followed by washing, the bound antigen on the solid phase is reacted with excess labeled antibody (the detection antibody). After further washing, the label is measured, and the signal of the label is directly correlated with the amount of antigen present. A modification of the sandwich-ELISA is the double sandwich-ELISA, which involves a third antibody. The third antibody carries a label and reacts with an unlabeled detection antibody already bound to the antigen. Then the signal of the label is measured, and like before, the signal of the bound label is directly correlated with the amount of the antigen present.
The advantages of sandwich-ELISA are that, by using a capture antibody on a solid phase, the method can enrich and purify an antigen from a fluid sample containing complicated components (this is especially important for the detection of a low abundance protein), and that, by using a pair of capture antibody and detection antibody, the double affinity bindings significantly increase the specificity of the detection. However, in this post-genomics era, to develop a sandwich-ELISA for a protein is still a big challenge for scientists. Why is it so difficult to develop a sandwich-ELISA? Because it is hard to find a capture antibody which maintains the antigen binding capacity after its immobilization on a solid phase, as about 80% of antibodies are denatured after immobilization on the solid phase; and it is even more hard to find a pair of matched capture antibody and detection antibody which form a sandwich complex with an antigen for the successful antigen capture and detection. All of these problems of sandwich-ELISA lead to the assay only can be used in limited number of proteins discovered.
Since Engvall and Perlmann published their first paper on ELISA in 1971 (Lequin 2005), there are many inventions and literatures in the field of immunoassay. However, most of these researches are focused on improvements of sandwich-ELISA, not worked on inventing an alternate convenient and robust immunoassay to overcome the technical difficulty of sandwich-ELISA, the requirement of a pair of perfect matched capture antibody and detection antibody for specific antigen detection and quantitation. For example, U.S. Pat. No. 5,236,849 worked on reducing the label signal background and increasing sensitivity of sandwich-ELISA by adding additional steps of dissociating the antigen-antibody complex from the solid phase and then rebinding the complex to a new affinity solid phase which attached with a reactive group which is capable of specifically binding with the complex; although U.S. Pat. No. 5,236,849 used two solid phases and a dissociating step in its patent claims, the invention does not solve the technical limitations of sandwich-ELISA by following facts: U.S. Pat. No. 5,236,849 uses sandwich complex mechanism of sandwich-ELISA for antigen or antibody's capture and detection; and in dissociating step of U.S. Pat. No. 5,236,849 the complex of antigen and antibody is dissociated from the solid phase in a form of the immuno-complex, not breaking the complex to elute un-complexed antigens into the liquid phase of the Dissociation buffer; in addition, both solid phases of U.S. Pat. No. 5,236,849 used are affinity binding solid phases (in which there is at least an affinity binding component immobilized), which means the method is not an universal method, as the setting of two different affinity binding solid phases for each antigen analysis is very difficult; finally, the detection step of U.S. Pat. No. 5,236,849 focuses on the detection of complex of antigen and antibody, not on the single antigen or single antibody, that is “assaying for the complex, bound to the second solid carrier.”(Copied from claim 1C of U.S. Pat. No. 5,236,849). Another example is the patent of U.S. Pat. No. 5,236,830; the inventor also used two solid phases and a dissociating step for antigen assay. But the goal of the invention is to increase the sensitivity of the antigen detection, and the mechanism of the antigen's capture and detection is sandwich-ELISA, and the two solid phases used are all affinity binding solid phases to increase detection sensitivity. Therefore, both U.S. Pat. No. 5,236,830 and U.S. Pat. No. 5,236,849 are not an invention which solves the technical challenge (the requirement of a pair of matched capture antibody and detection antibody for antibody-antigen-antibody complex formation) of sandwich-ELISA. In contradictory, they are a more complicated and two affinity solid phases based sandwich-ELISA, that need several pairs of matched capture antibodies and detection antibodies. Through careful literature searches, it seems all inventions of immunoassay before current invention have not solved the technical limitations of sandwich-ELISA successfully; specifically, they continue using sandwich-ELISA mechanism, the formation of antibody-antigen-antibody complex, to capture and detect/quantitate antigen. Thus, there is a need to invent a convenient and robust method to replace sandwich-ELISA with a novel alternate mechanism for the antigen capture and detection.
As mentioned earlier, to develop a convenient and robust method for analysis of protein-protein interaction is critically important. It is because the protein-protein interactions are at the center of almost every cellular process from cell motility, DNA replication, transcription, translation, splicing, secretion, cell cycle control, signal transduction, cell proliferation, to cell-cell interactions; and because classical and natural method for analyzing protein-protein interactions, the immunoprecipitation (IP), is not a high through put and not a convenient method, as it needs coupling with other assays such western blot and immuno-blotting to get final result. Therefore, there is a need to invent a convenient and robust immunoassay method to replace IP.
Another important immunoassay is immunohistochemistry (IHC) and immunocytochemistry (ICC). IHC is a technique developed by Coons six decades ago (Coons A H C H et al., 1941). Today IHC has a central role in the field of diagnostic medicine and research of pathology. IHC's variation ICC also has important role in biomedical research, clinical diagnostics, and drug discovery. Both techniques of IHC and ICC are using a detection antibody to detect an antigen immobilized on a solid phase. The major difference is that the antigen detected by IHC is located in tissue immobilized on a solid phase, while the antigen detected by ICC is located in cells fixed on a solid phase.
The major challenge of ICC and IHC is that it is difficult to set up quantitative ICC and IHC. Traditionally ICC and IHC are qualitative methods or semiquantitative methods (Kaczmarek E et al 2004, Lang et al 2006, Dodson 2002, Ramos-Vara 2005, Guardigli et al 2005). The presence and amount of the antigen on the tissue or cells immobilized on a solid phase is judged by assigning scores of a skilled observer such as pathologist via using a proper microscope, or by cellular or tissue image method that involves image capture and image data analysis. It is well known that the scores method is not objective; the results from the scores method for quantitation can be varied significantly with different observers. The image method also is not an ideal quantitative method as it is not a direct measurement of antigen quantity, needs expensive equipment, consists of complicated data manipulation procedures that might be leading to false positive, and is very time consuming. Thus, there is an urgent need to develop better method for quantitative ICC or quantitative IHC.
This invention discloses a novel immunoassay method to replace sandwich-ELISA for protein or other antigens' detection and quantitation, and to replace IP for protein-protein interaction analysis. The basic idea of the invention is that by separating the process of antigen capture and detection of sandwich-ELISA in a solid phase into two solid phases-based processes (one affinity binding solid phase and one non-affinity binding solid phase) to break the strict requirement of a perfect matched two antibodies for formation of sandwich antibody-antigen-antibody complex in sandwich-ELISA. The first solid phase (the affinity binding solid phase) of present invention is for the capture of the antigen by affinity binding, and the second solid phase (non-affinity binding solid phase) of present invention is for immobilizing the antigen for specific detection. As the invention need two solid phases and can be used to set up immunoassay for unlimited number of proteins, the invention is named as universal tandem solid-phases based immunoassay (UTSIA).
The invention also discloses an important variant of UTSIA, cell-based UTSIA, for quantitative IHC and ICC. It is a cell-based ELISA equivalent immunoassay which is used to detect and quantitate antigen of interest in cells or tissue immobilized on a solid phase. The advantage of the cell-based UTSIA comparing with cell-based ELISA is that it eliminates the background of cells or tissue of cell-based ELISA in the detection/quantitation step. The applications of the cell-based UTSIA comprise cell based or tissue based biomarker evaluation, detection, and quantitation, in the fields of biomedical research, clinical diagnosis, and drug discovery.